Population Density, Size Structure, and Reproductive Cycle Of

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Article Population Density, Size Structure, and Reproductive Cycle of the Comestible Sea Urchin Sphaerechinus granularis (Echinodermata: Echinoidea) in the Pagasitikos Gulf (Aegean Sea)

Dimitrios Vaﬁdis 1,*, Chryssanthi Antoniadou 2 and Vassiliki Ioannidi 1

1 Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, 38446 Volos, Greece; [email protected] 2 Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; [email protected] * Correspondence: dvaﬁ[email protected]; Tel.: +30-241-093-133

Received: 3 August 2020; Accepted: 21 August 2020; Published: 26 August 2020

Simple Summary: Regular urchins are dominant grazers in the shallow sublittoral seabed. Several species are edible and commercially harvested, among which Sphaerechinus granularis is of increasing commercial importance due to the depletion of the common urchin Paracentrotus lividus. As there are very few studies on the biology of the species, the present work examines the population density, size structure, and reproduction of S. granularis in the Aegean Sea (eastern Mediterranean). Population density, in-situ estimated along transects, conforms to previous reports for the Aegean Sea. Size-structure, estimated from 20 randomly collected specimens at monthly base, showed one mode at 65–70 mm or 70–75 mm, according to the sampling location. Sea urchins had larger dimensions in the sheltered site. The monthly variation of the gonad-somatic index and the histological analyses of the gonads, estimated from the same 20 randomly collected specimens (per month and station), showed that the species reproduces once each year, in spring. The results of the present study provides baseline knowledge on the biology of S. granularis and are important for the viable management of its developing ﬁshery.

Abstract: Sphaerechinus granularis is a common grazer that lives in various sublittoral habitats, displaying typical covering behavior; i.e., putts shell-fragments, pebbles, and algae on its test. It is an edible species of increasing commercial importance due to the depletion of the common urchin’s, Paracentrotus lividus, stocks. Its biology, however, is not adequately studied over its distributional range. The present study examines population density, size structure, and reproductive biology of S. granularis in the Aegean Sea. Samplings were made with SCUBA-diving (8–10 m) and included: (i) visual census along transects to estimate density, and (ii) random collection of specimens at monthly intervals to assess biometry and gametogenesis. Population density had moderate values that almost doubled when inputted to Distance software. S. granularis had larger dimensions in the sheltered site; size-structures were unimodal (65–70 mm and 70–75 mm, in exposed and sheltered site, respectively). An annual reproductive cycle was evident, according to GSI and gonads’ histology, with a clear spawning peak in early spring. This pattern conforms to previous reports from the Atlantic, but precedes those from the Mediterranean (reproduction in summer). The provided baseline knowledge on the biology of S. granularis is important for the viable management of its developing ﬁshery.

Keywords: population abundance; biometry; gonad-somatic index; histology; sea urchin; Sphaerechinus; Aegean Sea

Animals 2020, 10, 1506; doi:10.3390/ani10091506 www.mdpi.com/journal/animals Animals 2020, 10, x 2 of 12

1. Introduction The blunt or purple sea urchin, Sphaerechinus granularis (Lamarck 1816), is an edible species distributed in the NE Atlantic and the Mediterranean Sea [1]. The species lives in a variety of habitat Animalstypes2020 [2], 10in, 1506both hard [3] and soft bottoms covered by seagrass meadows [4] or on maërl beds [5]. Its2 of 12 bathymetric distribution expands from the intertidal down to 130 m depth [6], but it is most usually found in the shallow sublittoral (5 to 15 m) where it forms locally dense populations [7]. As other sea 1. Introductionurchins, it manifests a cryptic behavior (Figure 1) by occupying crevices and by camouflaging, as it coversThe bluntits test orwith purple shell fragments, sea urchin, pebbles,Sphaerechinus and algae granularis [8]. (Lamarck 1816), is an edible species The species is harvested for its gonads in France, Spain and in few other countries, since the distributed in the NE Atlantic and the Mediterranean Sea [1]. The species lives in a variety of habitat beginning of 1980s, although it is less appreciated than Paracentrotus lividus (Lamarck, 1816), the types [2] in both hard [3] and soft bottoms covered by seagrass meadows [4] or on maërl beds [5]. target species in sea urchins fishery [5]. In recent years, however, the depletion of P. lividus natural Its bathymetric distribution expands from the intertidal down to 130 m depth [6], but it is most usually stocks is shifting sea urchin fishery to alternative species such as S. granularis, which is a fast-growing, foundlarge-sized in the shallow urchin that sublittoral contains (5 voluminous to 15 m) where gonads it forms [5]. In locally the Hellenic dense populationsSeas, S. granularis [7]. As is locally other sea urchins,consumed, it manifests but it is a not cryptic officially behavior harvested, (Figure and1) byso, occupyingthe minimum crevices landing and size by of camouﬂaging, 7 cm [9] is not as it coversapplicable its test [7]. with Unfortunately, shell fragments, fisheries pebbles, data are and completely algae [8]. lacking for the region.

FigureFigure 1. Covering1. Covering behavior behavior of of the the sea sea urchinurchin SphaerechinusSphaerechinus granularis granularis: :underwater underwater photography photography of a of a specimenspecimen in in station station 2, 2, S2 S2 (photo (photo from from Dr. Dr. Alexios Alexios Lolas).Lolas).

TheDespite species the is increasing harvested importance for its gonads of S. ingranularis France, as Spain a fishery and inresource, few other its biology countries, is scarcely sincethe beginningstudied.of More 1980s, explicitly, although the it existing is less appreciatedinformation thanis limitedParacentrotus to the reproductive lividus (Lamarck, biology 1816),[1,5], and the the target specieslife strategy in sea urchinsof the species fishery in [relation5]. In recent to food years, availability however, [10] the in the depletion western of MediterraneanP. lividus natural and stocksthe is shiftingNE Atlantic sea (Brittany). urchin fishery In Brittany, to alternative the species species has a rather such brief as S. breedi granularisng period, which that begins is a fast-growing, in spring (April or May) and extends until early summer (June), in dependence to the annual thermal regime large-sized urchin that contains voluminous gonads [5]. In the Hellenic Seas, S. granularis is locally [5]. Seawater temperature affects mainly the onset of spawning, which exhibits inter-annual consumed, but it is not officially harvested, and so, the minimum landing size of 7 cm [9] is not variability in relation to this environmental factor [9]. In Mediterranean, the breeding period begins applicable [7]. Unfortunately, fisheries data are completely lacking for the region. in summer [1,11] but may extend until November [1]. It is widely accepted that the triggering factor initiatingDespite spawning the increasing is the rapid importance rise in temperature of S. granularis thatas usually a fishery occurs resource, in spring its in biology temperate is scarcely seas studied.[5,9,11], More although explicitly, differences the existing in food information quality are al isso limited involved to the[1]. reproductiveFor the Aegean biology Sea, there [1,5 ],are and no the liferelevant strategy studies of the other species than in a relation few reports to food on its availability distribution [10 [12]] in and the po westernpulation Mediterranean ecology [7]. and the NE AtlanticWithin (Brittany). this context, In Brittany, the present the work species aims has to a ratherstudy the brief population breeding density, periodthat the beginssize structure in spring (Apriland orthe May) reproductive and extends biology until of early S. granularis summer in (June), the central in dependence part of the Aegean to the annual Sea (Pagasitikos thermal regime Gulf). [5]. Seawater temperature affects mainly the onset of spawning, which exhibits inter-annual variability in relation2. Materials to this environmentaland Methods factor [9]. In Mediterranean, the breeding period begins in summer [1,11] but may extend until November [1]. It is widely accepted that the triggering factor initiating spawning is the2.1. rapidStudy Area rise inand temperature Field Sampling that usually occurs in spring in temperate seas [5,9,11], although differencesThe inpresent food qualitystudy was are carried also involved out in Pagasitikos [1]. For the Gulf, Aegean central Sea, Aegean there areSea, no which relevant is an studies enclosed, other thanshallow a few water, reports meso-oligotrophic on its distribution gulf [12 [13].] and Two population stations were ecology sampled: [7]. S1 (39°18′19.3″ N, 22°56′35.3″ E)Within and S2 this (39°18 context,′25.1″ the N, present 23°5′50.9 work″ E), aims located to study at the the north population and northeast density, theparts size ofstructure the Gulf, and therespectively reproductive (Figure biology 2). The of S. sea granularis bottom wasin the sandy central in both part stations, of the Aegean either unvegetated Sea (Pagasitikos or covered Gulf). by moderately dense seagrass meadows, with large boulders and scattered rocks in the shallower zone 2. Materials and Methods

2.1. Study Area and Field Sampling The present study was carried out in Pagasitikos Gulf, central Aegean Sea, which is an enclosed, shallow water, meso-oligotrophic gulf [13]. Two stations were sampled: S1 (39◦18019.300 N, 22◦56035.300 E) and S2 (39◦18025.100 N, 23◦5050.900 E), located at the north and northeast parts of the Gulf, respectively (Figure2). The sea bottom was sandy in both stations, either unvegetated or covered by moderately dense seagrass meadows, with large boulders and scattered rocks in the shallower zone Animals 2020, 10, x 3 of 12 Animals 2020, 10, 1506 3 of 12 (up to 5 m depth). Fleshy algae of the genus Cystoseira prevailed in S1 and coralline algae together with the brown alga Padina pavonica in S2. The above stations differ mainly on exposure level [14,15]. (up to 5 mSamplings depth). Fleshywere made algae in 2010 of the by genusscientistsCystoseira using SCUBAprevailed diving inat approximately S1 and coralline 8–10 algae m depth, together with theand brown included alga a combinationPadina pavonica of visualin S2.cens Theus and above random stations collection diﬀer of mainly specimens. on exposure level [14,15].

FigureFigure 2. Map 2. Map of theof the study study area area indicating indicating the loca locationtion of of sampling sampling stations stations (S1 and (S1 andS2). S2).

Samplings2.2. Population were Density made and in Size 2010 Structure by scientists using SCUBA diving at approximately 8–10 m depth, and includedThe aconventional combination distance of visual sampling census method, and random which models collection detection of specimens. probability as a function of distance from a line or point transect and assumes the detection of all individuals at zero distance 2.2. Population[16–18] was Density applied, and in Size January Structure 2010, to estimate the population density of S. granularis. Five (5) Thereplicate conventional 50-m line transects distance were sampling surveyed method, in each which station; models replicate detection transects probabilitywere randomly as aapplied function of but desisted at least 10 m apart to avoid repetition. All living S. granularis individuals found along distance from a line or point transect and assumes the detection of all individuals at zero distance [16–18] each random 50-m line transect were counted, and their perpendicular distance (d) from the line— was applied, in January 2010, to estimate the population density of S. granularis. Five (5) replicate 50-m under the assumption that d is negatively related with detection probability—within a 3 m belt line transectsastride to were the line surveyed transect in was each also station; recorded replicate with a transectstape-meter were and randomlyinputted to applied Distance but software desisted at least 10(version m apart 6, second to avoid release). repetition. Based Allon recorded living S. dist granularisances, aindividuals detection function found is along fitted each onto randomthe data 50-m line transectand used were to estimate counted, the anddetection their probability, perpendicular i.e., the distance proportion (d) from of non-detected the line—under individuals the assumption in the that dgiven is negatively area, and related to assess with the detection density of probability—within the studied population a 3 m [16–18]. belt astride Distance to the measurements line transect was also recordedshould be with accurately a tape-meter recorded and and inputtedthe organism to Distance of interest software should move (version slower 6, that second the observer release). to Based on recordedavoid biases. distances, Although a detection widely applied function in terrestrial is fitted ontoecology, the the data method and used has been to estimate scarcely theused detection for marine species other that mammals [19,20]. probability, i.e., the proportion of non-detected individuals in the given area, and to assess the density At each station, 20 sea urchin specimens were randomly collected at monthly intervals, from of the studied population [16–18]. Distance measurements should be accurately recorded and the January to December 2010, to estimate the size structure of the studied S. granularis population. organismConcurrently, of interest seawater should movetemperature, slower salinity, that the pH observer, and dissolved to avoid biases.oxygen Althoughwere recorded widely with applied an in terrestrialautographic ecology, conductivity-temperature-depth the method has been scarcely sensor, used CTD for (SeaBird marine electronics, species other Washington, that mammals DC, USA). [19 ,20]. At eachIn the station, laboratory, 20 the sea sea urchin urchin specimens specimens were were measured randomly for test collected diameter (Dt) at and monthly height intervals,(Ht) from Januaryusing an toelectronic December calliper 2010, (Mitutoyo to estimate Corporation, the size structureTakatsu Ward, of the Japan, studied 0.01 S.mm granularis precision),population. and Concurrently,drained for seawater 5-min on filter temperature, paper. Each salinity, specimen pH, was, and then, dissolved weighted oxygenfor total weight were recorded(tW) using withan an autographicelectronic conductivity-temperature-depth scale (0.001 g precision). sensor, CTD (SeaBird electronics, Washington, DC, USA). Analysis of variance, GLM ANOVA [21] was used to test for differences between the two In the laboratory, the sea urchin specimens were measured for test diameter (Dt) and height sampling stations in the estimated biometric variables (Dt, Ht, tW). Prior to the analyses, data were (Ht) using an electronic calliper (Mitutoyo Corporation, Takatsu Ward, Japan, 0.01 mm precision), tested for normality with the Anderson–Darling test, while the homogeneity of variance was tested and drainedwith Cohran’s for 5-min test. on The filter Fisher paper. LSD Each test specimenwas used was,for post-hoc then, weighted comparisons for totalwhen weight appropriate. (tW) using an electronicANOVAs scale were (0.001 performed g precision). using the SPSS software package (IBM SPSS statistics version 19). Analysis of variance, GLM ANOVA [21] was used to test for differences between the two sampling stations2.3. inReproductive the estimated Cycle biometric variables (Dt, Ht, tW). Prior to the analyses, data were tested for normalityThe monthly with the collected Anderson–Darling sea urchin specimens test, while from the both homogeneity stations were offurther variance processed was testedin the with Cohran’slaboratory test. Theto assess Fisher the LSD reproductive test was usedcycle forof the post-hoc species. comparisons More specifica whenlly, the appropriate. sea urchins were ANOVAs were performed using the SPSS software package (IBM SPSS statistics version 19).

2.3. Reproductive Cycle The monthly collected sea urchin specimens from both stations were further processed in the laboratory to assess the reproductive cycle of the species. More specifically, the sea urchins were Animals 2020, 10, 1506 4 of 12 dissected to remove and then, weigh the five gonads (Wg, 0.001 g precision). Weight measurements were used to calculate wet gonad-somatic index (GSI) as Wg/tW% to assess the reproductive condition of S. granularis [1]. GLM ANOVA was used to test for differences in GSI values between sampling stations (two-level random factor), months (12-level fixed factor), and of their interactions [21]. The specimens were sexed through the macroscopic examination of gonads or smear examination to calculate sex ratio ( / ), and the chi-square was apply to test for significant deviations from unity (1/1). The middle portion♂ ♀ of each individual’s gonads was fixed in formaldehyde, placed in cassettes and inputted in histokinette (Leica TP 1020, Leica Microsystems GmbH, Nussloch, Germany) for dehydration (immersion in ethanol solution of increasing concentrations), clearing (immersion in xylene solutions to replace ethanol with an organic dissolvent), and embedding in liquid paraffin wax. The gonadal tissue’ paraffin blocks were left for cooling (Leica EG 1150H, Leica Microsystems GmbH, Nussloch Germany) and then mounted on a microtome (Slee Mainz Cut 5062, SLEE medical GmbH, Mainz, Germany) for sectioning (5 µm sections). The sections were stained with the hematoxylin–eosin regressive staining procedure, covered with Canada balsam mounting medium, and observed under light microscopy (Axiostar plus Carl Zeiss optical microscope, Carl Zeiss Light Microscopy, Gottingen, Germany, connected with a ProgRes C10 digital camera, 10 100 magnification, JENOPTIC Optical Systems × GmbH, Jena, Germany) for the histological examination of gonads. The different developmental stages of gametogenesis were assessed according to the 6-level (I = recovery, II = growing, III = premature, IV = mature, V = pre-spent, VI = spent) Byrne’s classification scale [22], for both female and male urchins, photographed and processed through image analysis (ProgRes Capture Pro 2.1 software). This scale, originally developed to assess the sea urchin’s P. lividus gametogenesis, has been also successfully applied in S. granularis [5].

3. Results

3.1. Environmental Parameters The recorded environmental parameters generally showed similar values between the two sampling stations (Table1). Sea-bottom temperature at 8 m depth varied annually from 13.28 to 27.67 and from 13.17 to 27.12 in S1 and S2, respectively, following the seasonal pattern of atmospheric warming in the study area. Salinity decreased in summer and autumn in both stations, whereas between-month diﬀerences are also observed. Dissolved oxygen varied temporally between sites, being generally increased at the exposed S2 station, while pH varied around 8.28 with very small diﬀerences between stations and months.

Table 1. Monthly values of the recorded environmental parameters, temperature T (◦C), salinity S (psu) pH, and dissolved oxygen O2 (mg/L) in the sampling stations (S1 and S2) of Pagasitikos Gulf.

T S pH O2 Month/Parameter S1 S2 S1 S2 S1 S2 S1 S2 January 13.96 13.17 36.99 38.21 8.27 8.33 5.21 4.01 February 13.28 13.46 37.67 38.03 8.28 8.27 3.04 5.11 March 13.31 13.53 37.78 38.46 8.23 8.29 4.76 6.49 April 15.34 15.94 38.33 37.94 8.28 8.28 6.62 6.49 May 21.21 19.92 37.54 37.54 8.29 8.26 5.88 2.48 June 25.04 26.45 37.47 36.64 8.26 8.24 6.61 5.71 July 27.08 27.12 36.46 36.18 8.26 8.23 5.44 2.45 August 27.67 26.78 36.12 36.76 8.26 8.31 2.06 5.02 September 24.28 25.03 36.84 36.49 8.29 8.29 4.92 5.92 October 21.79 22.04 36.42 36.84 8.31 8.35 5.22 5.54 November 18.31 17.95 36.71 36.99 8.34 8.39 2.48 3.67 December 16.01 15.64 36.87 36.21 8.31 8.24 2.99 4.97 AnimalsAnimals2020 2020, 10,, 10 1506, x 5 5of of 12 12 Animals 2020, 10, x 5 of 12 3.2. Population Density and Size Structure 3.2. Population Density and Size Structure 3.2. PopulationOverall 76Density sea urchins and Size were Structure detected by visual census along the five replicate line-transects (41 Overall 76 sea urchins were detected by visual census along the five replicate line-transects observationsOverall 76 in sea S1 urchins and 35 were in S2).detected Mean by populati visual censuson density along (Table the five 2) replicate over both line-transects stations was (41 5 (41observationsindividuals/100 observations in inS1m S12and according and 35 35in in S2).to S2). originalMean Mean populati field population dataon density(mean density number (Table (Table 2)of over2sea) over urchinsboth both stations observed stations was and was 5 2 5individuals/100 individualscounted per/ 100transect); m m2 accordingaccording when these to to original data original are fieldinputted field data data to (mean Distance (mean number number software, of of seathe sea relevanturchins urchins valueobserved observed increased and and countedcountedto 8.5 perindividuals/100 per transect); transect); whenwhen m2. thesethesePopulation data are density inputted inputted differed to to Distance Distance between software, software, sampling the the relevantstations relevant value(p value < 0.05) increased increased being 2 totohigher 8.5 8.5 individuals individuals/100 in S1 (5.5/ 100or 9.2 mm individuals/1002.. Population density densitym2) compared differed differed to between betweenS2 (4.5 or sampling sampling7.8 individuals/100 stations stations (p (mp< 2<0.05)). 0.05) being being 2 2 higherhigher in in S1 S1 (5.5 (5.5 or or 9.2 9.2 individualsindividuals/100/100 m2)) compared compared to to S2 S2 (4.5 (4.5 or or 7.8 7.8 individuals/100 individuals/100 m m2). ). Table 2. Population density of Sphaerechinus granularis in S1 and S2 sampling stations of Pagasitikos TableTableGulf 2. according2.Population Population to original densitydensity field ofof SphaerechinusSphaerechinus and Distance granularis software indatain S1 S1 and and S2 S2 sampling sampling stations stations of ofPagasitikos Pagasitikos Gulf according to original field and Distance software data. Gulf according to original field andOriginal Distance Field software Data data Distance Software Data Station OriginalIndividuals/100Original Field Field Data Data m 2 DistanceIndividuals/100Distance Software Software Data m2 Data StationStation 2 2 S1 Individuals/100Individuals5.5 /100 m m2 Individuals/100Individuals 9.2 m/2100 m S1S1S2 5.54.5 5.5 9.2 7.8 9.2 S2MeanS2 value 4.5 5.04.5 7.8 8.5 7.8 Mean value 5.0 8.5 Mean value 5.0 8.5 In total 480 S. granularis specimens were processed; 240 from S1 and 240 from S2. At S1, mean sizeInIn was total total 69.63 480 480S. ±S. granularis7.87 granularis mm andspecimens specimens 45.03 ± were6.51 were mm processed; processed; for test 240diameter 240 from from S1 and S1 and and height, 240 240 from respectively.from S2. S2. At At S1, S1, In mean meanS2, the size wassizerelevant 69.63 was 69.63 sizes7.87 ±were mm 7.87 and 66.56mm 45.03 and± 8.27 45.036.51 mm ± mm and6.51 for42.37 mm test for± diameter8.13 test mm diameter for and Dt height, andand height,Ht. respectively. Mean respectively. weight In S2,was In the 152.463 S2, relevant the ± ± ± sizesrelevant47.176 were and sizes 66.56 136.722 were8.27 66.56± mm61.581, ± and8.27 in 42.37 mmS1 and and 8.13S2, 42.37 respectively. mm ± for8.13 Dt mm and for Ht. Dt Mean and Ht. weight Mean was weight 152.463 was 152.46347.176 and± ± ± ± 136.72247.176ANOVA and61.581, 136.722 results in S1± 61.581, and revealed S2, in respectively.S1 significant and S2, respectively. between-sta tions differences in the estimated biometric ± variablesANOVAANOVA (test results results diameter revealedrevealed Dt: F significant=significant 17.25 p < 0.01,between-sta between-stations test heighttions Ht: differencesdi F ff=erences 15.68 pin < in the0.01, the estimated total estimated weight biometric biometric tW: F = variablesvariables9.89 p < (test (test0.01); diameterdiam botheter size Dt: and F F = = biomass17.2517.25 p p< of <0.01, 0.01,S. granularistest test height height were Ht: Ht: Fmuch = F15.68= higher15.68 p < 0.01, pin< S1total0.01, (Figure weight total 3). weight tW: On F the = tW: F9.89=contrary,9.89 p

0.01). didifferencesﬀerences were detected in in the the urchin’s urchin’s biometry biometry between between sexes sexes (ANOVA (ANOVA results,results,p p> >0.01). 0.01). 71 46 160 70 45 155 71 46 160 69 150 44 70 45 155 68 145 150

69 43 tW(g) Ht (mm) Ht Dt (mm) 67 44 140 68 145 66 42 135 43 tW(g) Ht (mm) Ht Dt (mm) 67 140 41 130 65 42 66 S1 S2 S1 S2 135 S1 S2 65 41 130 Figure 3. MeanS1 size S2(test diameter Dt and heightS1 Ht in S2 mm) and biomass (totalS1 weight tW S2 in g) ± FigureFigureFisher 3. 3. LSDMean Mean of size thesize (testsea (test urchin diameter diameter Sphaerechinus Dt Dt and and height height granularis Ht Ht in mm)in in mm) S1 and and and biomass S2 biomass sampling (total (total weightstations weight tW of intWPagasitikos g) in g)Fisher ± ± LSDFisherGulf. of theLSD sea of urchinthe seaSphaerechinus urchin Sphaerechinus granularis granularisin S1 and in S2S1 samplingand S2 sampling stations stations of Pagasitikos of Pagasitikos Gulf. Gulf. SizeSize frequency frequency analysis, analysis, constructed constructed for Dt was was unimodal unimodal in in both both stations; stations; modal modal length length was was at at 70–75Size mm frequency in S1 and analysis, slightlyslightly constructed lower at for 65–70 Dt was mm unimodal in S2 (Figure(Figure in both 44).). stations; SizeSize frequencyfrequency modal length distributions was at temporally70–75temporally mm skewed in skewed S1 and (data (data slightly not not shown) shown) lower either eitherat 65–70 to to smaller smalmmler in (e.g., (e.g.,S2 (Figure July, August, 4). Size September) September) frequency or ordistributions larger larger modal modal lengthstemporallylengths (e.g., (e.g., skewed March, March, (data April). April). not shown) either to smaller (e.g., July, August, September) or larger modal lengths (e.g., March, April). 100 S1 S2

100 S1 S2Females 75 Males Females 75 Males 50

50 25 number of specimens number

25 number of specimens number 0 40-45 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90 90-95 95-100 40-45 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90 90-95 95-100 0 40-45 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90 90-95test 95-100 diameter40-45 (Dt 45-50 mm) 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90 90-95 95-100 Figure 4. Size frequency histogram (Dt in mm)test diameter of the (Dt sea mm) urchin Sphaerechinus granularin in S1 and S2 sampling stations of Pagasitikos Gulf. Animals 2020, 10, x 6 of 12

Figure 4. Size frequency histogram (Dt in mm) of the sea urchin Sphaerechinus granularin in S1 and S2 Animals 2020, 10, 1506 6 of 12 sampling stations of Pagasitikos Gulf.

3.3.3.3. Reproductive Reproductive Cycle Cycle Overall,Overall, 265 S. granularis specimensspecimens werewere females females and and 215 215 males males (S1: (S1: 139 139and♀ and 101 101, S2:♂, S2: 126 126and♀ 2 and114 114in♂ S2). in TheS2). sexThe ratio sex ratio was significantlywas significantly biased biased in favour in favour of females of females in S1 in (S1:♀ S1 (S1:/ = ♂0.72,/♀ = x0.72,2 =♀6.017 x = 2 6.017p <♂0.05), p < 0.05), whereas whereas no significant no significant deviation deviation from from unity unity was detectedwas detected in S2 in (S2: S2 ♂(S2:/♀= ♂0.90/♀ = x 20.90= 0.043 x = 0.043p > 0.05), p > 0.05), being being about about 1:1.233 1:1.233 over over the the entire entire sea sea urchin urchin population populationstudied, studied,♂ suggestingsuggesting♀ a slight slight prevalenceprevalence of of females females in in Pagasitikos Pagasitikos Gulf. GSIGSI ranged ranged from from 0.316 0.316 (September (September 2010) 2010) to 3.264 3.264 (March (March 2010) with a mean value at 1.980 ± 1.3011.301 ± atat S1 S1 and and 2.442 2.442 ± 1.1631.163 at at S2. S2. GSI GSI showed showed sign significantificant differences differences between between stations stations (F = 16.6216.62 pp << 0.01),0.01), ± withwith increased valuesvalues in in S2. S2. The The relevant relevant seasonal seasonal diff differenceserences were were also also significant significant (F = 12.91(F = p12.91< 0.001). p < 0.001).In both In stations, both stations, increased increased GSI values GSI values were were recorded recorded from from late late winter winter to early to early spring, spring, in in March March in inparticular, particular, whereas whereas a secondary a secondary rise rise was was also also observed observed in July in July (Figure (Figure5). The 5). The seasonal seasonal GSI trendGSI trend was wasnegatively negatively correlated correlated with with the annual the annual sea-water sea-water temperature, temperature, especially especially in S1 (Sin% S1= (S0.81ρ = and−0.81 and0.34, − − −for0.34, S1 for and S1 S2, and respectively), S2, respectively), whereas whereas non-significant non-significant correlations correlations are found are with found the otherwith measuredthe other measuredenvironmental environmental variables. variables.

S1 3.9

2.9

1.9

0.9

-0.1 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

GSI S2 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec FigureFigure 5. TemporalTemporal trend trend of of the the mean mean gonad-somatic gonad-somatic index index GSI GSIFisher ± LSDFisher of SphaerechinusLSD of Sphaerechinus granularis ± granularisat the two at sampling the two sampling stations (S1, stations S2) in (S1, Pagasitikos S2) in Pagasitikos Gulf. Gulf.

TheThe pattern pattern of ovarian growth waswas divideddivided intointo sixsix stagesstages (Figure (Figure6 ).6). Recovering Recovering ovaries ovaries (Stage (Stage I) I)are are filled filled with with nutritive nutritive phagocytes phagocytes together together with with some some small, small, previtellogenic previtellogenic oocytes oocytes and few and primary few primaryoocytes. Atoocytes. the next At growing the next stage growing (Stage stage II), early (Stage vitellogenic II), early oocytes vitellogenic appear. oocytes At the prematureappear. At stage the premature(Stage III) bothstage small (Stage and III) large both oocytes small and are large present, oocyt althoughes are present, they are although much less they in number are much that less small in numberones cohort. that small In the ones mature cohort. stage In (Stagethe mature IV) the stage ovaries (Stage are IV) filled the withovaries more are closely filled with packed more and closely large packedova (larger and oocyteslarge ova clearly (larger prevailed oocytes clearly in ova), prevai whereasled thein ova), germinal whereas epithelium the germinal still contains epithelium oogonia. still containsIn the pre-spent oogonia. stage In the (Stage pre-spent V), small stage oocytes (Stage andV), small nutritive oocytes phagocytes and nutritive are no phagocytes longer observable, are no longerwhereas observable, the ova further whereas increased the ova filling further in-between increase gaps.d filling However, in-between some gaps. gaps However, may be present some due gaps to mayspawn, be present which willdue beto furtherspawn, increasewhich will at thebe further spent stage increase (Stage at the VI) spent where stage only (Stage few relict VI) where oocytes only are fewpresent relict in oocytes an otherwise are present empty in gonad. an otherwise empty gonad. Animals 2020, 10, x 7 of 12

The pattern of testis growth was also divided into six stages (Figure 7). Recovering testes (Stage I) contained only nutritive phagocytes. In growing testes (Stage II), spermatids and spermatocytes are observable; they will be further developed to spermatozoans in premature testes (Stage III), which will fill the testis in the mature stage (Stage IV). In pre-spent stage (Stage V) spermatozoans are denselyAnimals 2020 packed, 10, 1506 in the testis, whereas in the spent stage (Stage VI) only few relict spermatozoans7 ofare 12 observedAnimals 2020 in, 10 the, x testis’ lumen. 7 of 12

The pattern of testis growth was also divided into six stages (Figure 7). Recovering testes (Stage I) contained only nutritive phagocytes. In growing testes (Stage II), spermatids and spermatocytes are observable; they will be further developed to spermatozoans in premature testes (Stage III), which will fill the testis in the mature stage (Stage IV). In pre-spent stage (Stage V) spermatozoans are densely packed in the testis, whereas in the spent stage (Stage VI) only few relict spermatozoans are observed in the testis’ lumen.

FigureFigure 6. 6. HistologyHistology of of ovaries ovaries of of SphaerechinusSphaerechinus granularis granularis.. NP NP == nutritivenutritive phagocytes, phagocytes, Og Og == oogenesis,oogenesis, RORO == relictrelict oocytes, oocytes, PO PO == previtellogenicprevitellogenic oocytes, oocytes, O O == oocytes,oocytes, OW OW == ovarianovarian wall, wall, GE GE = germinalgerminal epithelium,epithelium, N == nucleus,nucleus, Stage Stage I =I recovery,= recovery, Stage Stage II = IIgrowing, = growing, Stage Stage III= premature, III= premature, Stage IVStage= mature, IV = mature,Stage V Stage= pre-spent, V = pre-spen Staget, VI Stage= spent. VI = spent.

The pattern of testis growth was also divided into six stages (Figure7). Recovering testes (Stage I) contained only nutritive phagocytes. In growing testes (Stage II), spermatids and spermatocytes are observable;Figure 6. they Histology will be of further ovaries developedof Sphaerechinus to spermatozoans granularis. NP = in nutritive premature phagocytes, testes (Stage Og = oogenesis, III), which will ﬁll theRO testis = relict in oocytes, the mature PO = stage previtellogenic (Stage IV). oocytes, In pre-spent O = oocytes, stage OW (Stage = ovarian V) spermatozoans wall, GE = germinal are densely packedepithelium, in the testis, N = nucleus, whereas Stage in the I spent= recovery, stage Stage (Stage II VI)= growing, only few Stage relict III= spermatozoans premature, Stage are IV observed = in themature, testis’ Stage lumen. V = pre-spent, Stage VI = spent.

Figure 7. Histology of testes (right) of Sphaerechinus granularis. NP = nutritive phagocytes, L = lumen, Sc = spermatocytes, St = spermatids, GE = germinal epithethelium, TW = teste wall, RS = relict spermatozoa (stages are defined as in Figure 6).

The relative frequencies of the maturity stages in female and male urchins, made for each station separately, showed a synchronized reproductive pattern between sexes (Figure 8). A clear annual reproductive cycle was evident, with a spawning peak in spring (March and April). Recovering FigureFigure 7. 7. HistologyHistology of of testes testes (right) (right) of of SphaerechinusSphaerechinus granularis .. NP NP == nutritivenutritive phagocytes, phagocytes, L L == lumen,lumen, ScSc == spermatocytes,spermatocytes, St St == spermatids,spermatids, GE GE == germingerminalal epithethelium, epithethelium, TW TW == testeteste wall, wall, RS RS = relictrelict spermatozoaspermatozoa (stages (stages are are defined deﬁned as as in in Figure Figure 66).).

TheThe relative relative frequencies frequencies of of the the maturity maturity stages stages in in female female and and male male urchins, urchins, made made for for each each station station separately,separately, showed showed a a synchronized reproductive pa patternttern between between sexes sexes (Figure (Figure 88).). AA clearclear annualannual reproductive cycle was evident, with a spawning peak in spring (March and April). Recovering Animals 2020, 10, 1506 8 of 12

Animals 2020, 10, x 8 of 12 reproductive cycle was evident, with a spawning peak in spring (March and April). Recovering gonads were observed in almost all months, but theythey clearlyclearly prevailedprevailed duringduring summer.summer. Although small didifferencesﬀerences between between the the two two stations stations existed, existed, such such as the as presence the presence of immature of immature males in males February in Februaryin S2, the in overall S2, the annual overall reproductive annual reproductive pattern of pattern the species of the was species similar. was similar.

Stage VI Stage V Stage IV Stage III Stage II Stage I S1 S2 100

50 females 25

100

50 males

0 Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov Dec Figure 8. TemporalTemporal trend of the relative frequency of each gonadal developmental stage of female and male Sphaerechinus granularis individuals at the two sampling stations (S1, S2) in Pagasitikos Pagasitikos Gulf (I = recovery,recovery, II = growing,growing, III= III= premature,premature, IV IV == mature,mature, V V == pre-spent,pre-spent, VI VI == spent).spent).

4. Discussion Discussion Sphaerechinus granularis is a very common sea urchin inhabiting various sublittoral habitats and following specific specific depth-distributional patterns patterns in in different different geographic sectors according to local environmental conditions [7]. [7]. In In horizontally or orientediented mixed soft bottoms—consisting of seagrass meadows, muddy muddy sands, sands, and and scattered scattered boulders boulders covered covered by various by various algal algalspecies, species, as the asstudy the area— study thearea—the species species thrivesthrives around around 10 m. In 10 the m. Mediterranea In the Mediterraneann Sea, according Sea, according to the very to the few very relevant few relevant studies 2 conductedstudies conducted for S. granularis for S. granularis, its density, its ranges density from ranges 1.8 to from 315 individuals/100 1.8 to 315 individuals m−2 in /the100 NW m− (Spain)in the andNW (Spain)SW (Algeria) and SW sectors, (Algeria) respectively sectors, respectively [9,23]. In [9the,23 ].Aegean In the AegeanSea, an Sea, overall an overall density density of 4.8 of 2 individuals/1004.8 individuals/100 m−2 mhas− beenhas been reported reported -a value -a value very very close close to the to one the reported one reported in the in present the present study study (5.0 2 (5.0individuals/100 individuals /m100−2)—which, m− )—which, however, however, varied varied greatly greatly between between geographic geographic sectors, sectors, being much being lower much inlower the oligotrophic in the oligotrophic southern southern part [7]. part Such [7]. a Suchpattern a pattern has been has reported been reported as well asfor well Paracentrotus for Paracentrotus lividus andlividus attributedand attributed to the reduced to the reduced algal coverage algal coverage of the ofrocky the rockyshore shoreand accordingly and accordingly to limited to limited food supplyfood supply [7]. Pagasitikos [7]. Pagasitikos Gulf Gulf is characterized is characterized as asmeso-oligotrophic, meso-oligotrophic, with with much much increased increased nutrient nutrient inputs in its inner inner part, part, where where S1 S1 is is located located [13]. [13]. Therefore, Therefore, the the increased increased trophic trophic status status of of the the sheltered sheltered and innermost site may provide additional food supply to sustain aa denserdenser population.population. All previous density reports for the species were basedbased onon originaloriginal fieldfield data.data. However, by inputting these data data to to Density Density software—simulating software—simulating the the probability probability of of the the species species to to be be overlooked– overlooked– population densitydensity almostalmost doubled.doubled. This This suggest suggest an an underestimation underestimation of of density density values values when when based based on ondirect direct field field count count data, withdata, important with important implications implications for the management for the management of the species. of Accordingly,the species. Accordingly,the conservation the statusconservation of the sea status urchin of the may sea have urch beenin may favored have as been the populationfavored as the size population may have been size maylarger have than been originally larger judged. than originally According judged. to distance Acco samplingrding to distance density estimations,sampling density biases estimations, are typically biasesdue to are the movementtypically due of theto the target movement species or of random the target errors species [18,24 or]. AsrandomS. granularis errors is[18,24]. a sedentary As S. granularis is a sedentary species, its motility patterns cannot bias density estimations (i.e., moving faster than the observer does), and so, relevant differences are most probably related with its cryptic behavior, i.e., active covering of the test with shells, pebbles, and algae [8]. The possibility of Animals 2020, 10, 1506 9 of 12 species, its motility patterns cannot bias density estimations (i.e., moving faster than the observer does), and so, relevant differences are most probably related with its cryptic behavior, i.e., active covering of the test with shells, pebbles, and algae [8]. The possibility of overlooking those well-hidden sea urchin individuals during underwater surveys, especially in vegetated bottoms, is increasing. Accordingly, the implementation of a distance sampling method that uses a detection function fitted to the set of records to estimate the proportion of missed animals and correct density estimates seems to be more accurate for predicting the abundance of such species. Considering the size of the studied S. granularis population, significant differences were detected between the two sampling stations. According to the size spectra distributions, one mode at 70–75 mm was detected in S1, which is higher than the minimum landing size for the species (70 mm); in S2, the mode falls to 65–70 mm. Although the number of specimens was relatively low (20 specimens per month and station), this temporal trend, which conforms between the two stations, may be considered as indicative for the studied S. granularis population. GSI followed the opposite trend, with higher values in the exposed S2 station. The different environmental conditions—excluding temperature that showed very similar values between the two stations—may have favored the growth of S. granularis in the shelter and organically rich S1 station [14], as previously reported for other sea urchins [15]. S. granularis is a grazing species with typical herbivorous habits. It prefers feeding on decaying plant material and epiphytic algae, as well as on the rhizomes and roots of seagrasses or encrusting coralline algae [25]. It is considered as generalist feeder, consuming different food items according to their availability [26], and so, manifesting high phenotypic plasticity according to local environmental conditions, and especially food availability. The reduced hydrodynamics in S1 [13] resulting in increased nutrient supply and greater development of algal coverage (fleshy Cystoseira), may have forced resource allocation to somatic growth [10], in contrast to the greater investment to gonadal growth in S2. A similar population phenotypic response to different hydrodynamic conditions has been previously suggested for the species [27], as well as reduced somatic growth under food-limited conditions [28]. Mean gonadal weight of S. granularis was similar between the studied sites, and accordingly, the significantly reduced mean size of sea urchins in the exposed site may have caused the relevant rise in GSI. In March, where GSI picked in both sites, the collected sea urchins had reduced size and increased gonad weight in the exposed site, whereas the sex ratio was almost equal to unity in both sites. These results suggest the investment in gonadal growth under high hydrodynamics and probably less favored food supply. Differences in resource allocation have been reported for S. granularis [1], as well as for P. lividus. For the latter species, allocation of energy to somatic growth at the expense of reproduction has been reported from shelter sites, to explain size differences between sheltered and exposed sites [29]. Alternatively, illegal collection of larger urchins for local consumption from S2—S1 is restricted for fisheries—may explain the observed differences in the size-structure of the species. Sphaerechinus granularis is a gonochoristic species with external fertilization. Its size at sexual maturity varies around 5 cm in test diameter [5]. The vast majority of the collected urchins were larger than 5 cm in diameter, and so, GSI and histological analysis are reliable to describe its reproductive biology in the study area. Females predominate in S1, whereas both sexes were equally represented in S2. A sex ratio of 3:2 or 3:3 has been reported for the species [1], whereas a bias in sex ratio balance in favor of females seems to be common in other sea urchin species [30,31]. Literature data on the reproductive biology of S. granularis are limited. In the Atlantic (Brittany), the species is reported to have an annual reproductive cycle with a single spawning season in spring [5,9]. In the Mediterranean Sea, two spawning events have been reported to occur along the French coasts (Nice), a minor one in late spring and a major one in late summer [11]. This contradicts to another study from the Alboran Sea that reports a single spawning episode in summer that may last until autumn, depending on prevalent environmental conditions at the studied locations, whereas mature gonads may be present all year round [1]. In the present study, a clear major spawning event in spring (March–April) was documented based on both the GSI and histology. GSI dropped a month earlier in Animals 2020, 10, 1506 10 of 12

S2 but remained in an overall higher state in this station, resulting probably from the smaller size of sea urchins, as previously discussed. The seasonal GSI pattern in the sheltered S1 conforms better to relevant trends from Brittany populations [9], and partly to western Mediterranean ones where the maxima are observed in summer [1]. However, according to GSI, a second minor spawning event in early autumn (September) may be inferred for the species, which, however was not confirmed by the histological examination of the gonads. Environmental factors—such as food availability, diet quality, and hydrodynamics—may affect GSI without initiating gametogenesis [32]. A second rise in GSI has been observed in both Atlantic [9] and Mediterranean [1] S. granularis populations, explained by nutritional factors that enhance gonad size, since histological data rejected the possibility of a second spawning event. Therefore, GSI results should be cautionary interpreted when describing the reproductive biology of sea urchins. The results of the present study provide baseline knowledge on the biology of S. granularis in the Aegean Sea, and the eastern Mediterranean, where no previous information exists. The revealed plasticity in population parameters, even in closely located areas, may have serious implications for the species protection, by considering regulative measures based on local-scale knowledge (e.g., exact reproductive period, growth period) to viable manage its developing fishery. Moreover, as sea urchins’ metabolism is highly affected by climate change initiating increased energetic cost for the species survivorship and reproduction [33], recent comparative studies on the species biology are needed to predict future trends.

Author Contributions: Conceptualization, D.V.; Methodology, D.V.; Software, C.A. and V.I.; Validation, D.V., C.A. and V.I.; Formal analysis, C.A. and V.I.; Investigation, V.I.; Resources, D.V.; Data curation, V.I.; Writing—original draft preparation, C.A. and V.I.; Writing—review and editing, C.A. and D.V.; Visualization, C.A.; Supervision, D.V.; Project administration, D.V.; Funding acquisition, D.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to thank Alexios Lolas for his help during the sampling. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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